Lung transplantation is performed in over 3,000 persons annually who would otherwise likely die of end-stage lung diseases. Clinical outcomes are improving after lung transplantation, but these outcomes lag behind other solid organ transplants. Primary graft dysfunction (PGD) is a form of lung ischemia-reperfusion injury that occurs in the immediate post-transplant period and is associated with substantial early morbidity and mortality and subsequent chronic allograft rejection. A better fundamental understanding of the pathogenesis of PGD after lung transplantation is needed to identify novel pathways to inform new therapeutic approaches. Neutrophils are prominently recruited to the lung during ischemia-reperfusion injury, and recently neutrophils have been observed to release into the extracellular space their chromatin decorated with granular proteins- structures termed neutrophil extracellular traps (NETs). We hypothesize that NETs are formed in lung ischemia-reperfusion and are directly responsible for lung barrier disruption leading to PGD. We will test this hypothesis using state-of-the art mouse modeling, real-time tracking of immune events with lung intravital microscopy, and the use of biological samples from lung transplant recipients with and without PGD. In Aim 1, we will determine the spatial and temporal formation of NETs in a mouse orthotopic, single-lung transplantation model of PGD. We will define the NET trigger by focusing on activated platelets and also damage-associated molecular patterns that are released from the ischemic lung and then test therapeutic strategies related to these pathways. In Aim 2, we will determine the pathogenicity of NETs in the PGD model by testing mice that are incapable of producing NETs (PAD4-/-) and also mice that have excessive NET accumulation (DNase1-/-). We will target components of NETs (extracellular histones, neutrophil proteases) that might be responsible for NET-mediated lung toxicity. In Aim 3, we will use a prospective cohort study of human lung transplant recipients to test biological samples obtained post-transplantation in subjects with and without PGD. We will determine the presence of NETs and NET-triggers in plasma and bronchoalveolar lavage fluid and test for their association with PGD. We will also determine the in vivo regulation of NETs by DNase1 activity and the influence on PGD severity. These experimental and translational studies will provide definitive evidence on the role of NETs in PGD and will set the stage for future clinical trials for a condition-PGD-that has no effective therapies.